Nucleotide sequences encoding peroxisome-associated polypeptides, and their uses in the diagnosis and/or treatment of lung injuries and diseases, and of oxidative stress-related disorders

ABSTRACT

The present invention is related to an isolated and purified peroxisome-associated polypeptide. The present invention is also related to the nucleotide sequence encoding said amino sequence, the inhibitor directed against said sequences and their use in the diagnosis, treatment, and/or prevention of lung injuries or diseases and oxidative stress-related disorders.

This U.S. National Phase application claims priority under 35 U.S.C.§371 of International Application PCT/BE98/00124, filed Aug. 20, 1998,which claims priority of Belgian application BE 9700692, filed Aug. 20,1997.

FIELD OF THE INVENTION

The present invention is related to a new peroxisome-associatedpolypeptide, the nucleotide sequence encoding said polypeptide andportions thereof as well as their uses in the diagnosis of severaldiseases, especially the diagnosis and/or the treatment of lung injuriesand diseases, and of oxidative stress-related disorders.

BACKGROUND OF THE INVENTION

The peroxisomes are organelles nearly ubiquitous in eukaryotic cells.They contain enzymes essential for various catabolic and anabolicpathways. Some of these enzymes are expressed constitutively whileothers can be induced under appropriate conditions. Peroxisomes carryout a variety of essential reactions such as peroxisomal oxidation andrespiration, fatty acid beta-oxidation, cholesterol and dolicholmetabolism, ether-phospholipid synthesis, and glyoxylate and pipecolicacid metabolism.

The peroxisomal respiratory pathway is based upon the formation ofhydrogen peroxide by a collection of oxidases and the decomposition ofthe H₂O₂ by catalase These reactions are responsible for 20% of oxygenconsumption in liver, and several oxidases have been identified inperoxisomes. Ethanol elimination via catalase in peroxisomes may besignificant in addition to the oxidation via cytosolic alcoholdehydrogenase.

The peroxisomal beta-oxidation system catalyses the beta-oxidative chainshortening of a specific set of compounds which can not be handled bymitochondria: very long chain fatty acids, di- andtrihydroxycholestanoic acids, pristanic acid, long chain dicarboxylicacids, several prostaglandins, several leukotrienes, 12- and15-hydroxyeicosatetraeonic acid, and several mono- and polyunsaturatedfatty acids, which are of direct diagnostic relevance for someperoxisomal disorders.

Peroxisomes play also a major role in the synthesis of cholesterol andother isoprenoids. Fibroblasts from patients affected by disorders ofperoxisome biogenesis show low capacity to synthesise cholesterol.

Two enzyme activities responsible for introduction of the characteristicether linkage in ether-linked phospholipids (dihydroacetonephosphateacyltransferase (DHAPAT) and alkyldihydroxyacetonephosphate synthase(alkyl-DHAP synthase)) are localised in peroxisomes. These enzymes arenot yet cloned. As demonstrated by the identification of patients withdeficiency of either DHAPAT or alkyl-DHAP synthase with severe clinicalabnormalities, ether-phospholipids are of major importance in humans.

Peroxisomes are able to detoxify glyoxylate via alanine/glyoxylateaminotransferase. The deficiency of this cloned enzyme causeshyperoxaluria type I. L-pipecolate is a minor metabolite of L-lysine andis catabolised by the L-pipecolate oxidase localised in peroxisomes. Theenzyme is deficient in cerebro-hepato-renal (Zellweger) syndrome.

In human, the importance of peroxisomes was emphasised by a number ofinherited diseases involving either a defect in the biogenesis ofperoxisomes or a deficiency of one (or more) peroxisomal enzymes. Sofar, 12 different peroxisomal disorders have been described and most ofthem are lethal.

A wide variety of chemicals have been showed to produce peroxisomeproliferation and induction of peroxisomal and microsomal fattyacids-oxidising enzymes activities in rats and mice. Several peroxisomesproliferators have been shown to increase the incidence of liver tumoursin these species. Proposed mechanisms of liver tumour formation byperoxisomes proliferators include induction of sustained oxidativestress.

Therefore, newly identified molecules associated with peroxisomes couldbe used for the development of diagnostic tools and possibly for theimprovement of several therapeutical applications of various diseasesassociated with peroxisomal disorders. In addition, it is useful toidentify the molecules present in specific organs like the lung andwhich may be used as specific markers of inflammatory diseases as wellas lung injuries or diseases.

SUMMARY OF THE INVENTION

The Inventors have isolated and purified a new sequence of a lowmolecular weight human broncho-alveolar polypeptide. Said mammal,preferably human, protein or polypeptide (hereafter identified as B18humprotein) has been sequenced and its corresponding genomic DNA (SEQ ID NO8) and cDNA (SEQ ID NO 1) have been identified. Similarly, thecorresponding nucleotide and amino acid sequence from a rat (SEQ ID NO 3and 4) and from a mouse (SEQ ID NO 5 and 6) have been obtained.

Said sequences present several homologies with other peroxisomalproteins of yeast and comprise a carboxy-terminal tripeptide SQL whichis necessary for the specific targeting and translocation of severalproteins into the peroxisome.

Therefore, the present invention is related to a new isolated andpurified polypeptide sequence having a amino acid sequence whichpresents more than 70% homology, advantageously more than 85% homology,more preferably more than 95% homology, with the amino acid sequence SEQID NO 2. Said amino acid sequence is advantageously obtained from amammal, preferably from a rat, a mouse or a human.

The present invention is also related to the isolated and purifiedpolypeptide sequence corresponding to the amino acid sequence SEQ ID NO2 or a portion thereof, preferably an immunoreactive portion (putativeimmunogenic domain or T or B cell epitopes).

Said portions are advantageously comprised between:

Glutamic acid position 14—Glutamic acid position 28

Alanine position 27—Leucine position 37

Alanine position 43—Glutamic acid position 58

Glutamic acid position 58—Valine position 70

Valine position 81—Leucine position 98

Arginine position 96—Leucine position 113

Serine position 119—Serine position 130

Valine position 138—Threonine position 151

Preferably, said portion has more than 10, 20, 30, 50 or 70 amino acids.Specific portions of the amino acid sequence SEQ ID NO 2 are alsoportions of more than 70 amino acids which present at least 80% of theproteinic activity (see example 5) of the complete SEQ ID NO 2 sequence.Therefore, the amino acid sequence according to the invention can bepartially deleted while maintaining its activity, preferably itsanti-oxidative activity, which will be described hereafter.

According to the invention, the amino acid sequence SEQ ID NO 2 presentsa pI of 7.16 and a molecular weight of 17047 Dalton as hereafter definedby bidimensional electrophoresis.

The present invention is also related to the nucleotide sequenceencoding the amino acid sequence according to the invention and itsregulatory sequences upstream said coding sequence. A nucleotidesequence encoding the polypeptide according to the invention is agenomic DNA (see SEQ ID NO 10), a cDNA (see SEQ ID NO 1) or a mRNA,possibly comprising said upstream regulatory sequence. Advantageously,said nucleotide sequence presents more than 70%, advantageously morethan 85%, more preferably more than 95% homology with SEQ ID NO 1 or itscomplementary strand.

According to a preferred embodiment of the present invention, saidnucleotide sequence corresponds to the nucleotide sequence SEQ ID NO 1,its complementary strand or a portion thereof.

“A portion of the nucleotide sequence SEQ ID NO 1” means any nucleotidesequence of more than 15 base pairs (such as a primer, a probe or anantisense nucleotide sequence) which allow the specific identification,reconstitution or blocking of the complete nucleotide sequence SEQ ID NO1 (including its regulatory sequences upstream the coding sequence).

Said portions allow the specific identification, reconstitution orblocking by specific hybridisation with the nucleotidic sequence SEQ IDNO 1, preferably under standard stringent conditions, with sequenceslike probes or primers possibly labelled with a compound (radioactivecompound, enzyme, fluorescent marker, etc.), and can be used in aspecific diagnostic or dosage method like probe hybridisation (seeSambrook et al., §§9.47-9.51 in Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, N.Y. (1989)),genetic amplification (like PCR (U.S. Pat. No. 4,683,195), LCR (Wu etal., Genomics 4, pp. 560-569), CPR (U.S. Pat. No. 5,011,769)).

Exemplary stringent hybridisation conditions are as follows:hybridisation at 42° C. in 50% formamide 5×SSC, 20 mM sodium phosphate,pH 6.8 washing in 0.2×SSC at 55° C. It is understood by those skilled inthe art that variation of these conditions occur based on the length andGC nucleotide content of the sequence to be hybridised. Formulasstandard in the art are appropriated for determining exact hybridisationconditions (see Sambrook et al.

Preferred examples of said nucleotide portions are as follows:

! Sequence? Position 5′-gccatcccagcagtggaggtgtttg-3′ (SEQ ID 217-241 NO11) 5′-ttgaacagctctgccaggttcacc-3′ (SEQ ID 261-284 NO 12)5′-tggaggtgtttgaaggggagccag-3′ (SEQ ID 230-253 NO 13)5′-caggttcaccttgttccctggctc-3′ (SEQ ID 247-270 NO 14)5′-gggtatgggactagctggcg-3′ (SEQ ID 33-52 NO 15)5′-ctggccaacattccaattgcag-3′ (SEQ ID 747-768 NO 16)

and the sequences of respectively 601 (SEQ ID NO 8), 604 (SEQ ID NO 9)and 469 (SEQ ID NO 7) base pairs corresponding to specific mRNAalternative splicing of the B18 human nucleotide sequence as describedin FIG. 4 (the known genomic sequence incorporating several introns andexons is represented in the sequence SEQ ID NO 10).

Said sequences may be used for a genetic amplification or a probehybridisation as above-described.

The present invention is also related to a vector comprising thenecessary elements for the injection, transfection or transduction ofcells and having incorporated one or more of the nucleotide sequencesaccording to the invention. The vector according to the invention isselected from the group consisting of viruses, plasmids, phagemides,cationic vesicles, liposomes or a mixture thereof. Said vector maycomprise also one or more adjacent regulatory sequences (such aspromoter(s), secretion and termination signal sequence(s)),advantageously operably linked to the nucleotide sequence according tothe invention.

The present invention is also related to the cell transformed by saidvector and expressing the polypeptide according to the invention.

The nucleotide sequence according to the invention can be alsointroduced in said cell by the formation of CaPO₄-nucleic acidprecipitate, DEAE-dextran-nucleic acid complex or by electroporation.

Another aspect of the present invention is related to an inhibitor ofthe polypeptide according to the invention or the nucleotide sequenceaccording to the invention (including the upstream sequences likepromoter-operator regulatory sequence which may be inhibited by a cis-and/or transactivating repressor) . Said inhibitor is advantageously anantibody or a fragment of said antibody such as an hypervariable portionof said antibody directed against the amino acid or nucleotide sequenceof the polypeptide according to the invention. Other examples ofinhibitors according to the invention are antisense nucleotide sequenceswhich allow the blocking of the expression of the nucleotide sequenceaccording to the invention.

Another aspect of the present invention is related to a diagnosticdevice (such as a diagnostic kit or a chromatographic column) comprisingan element selected from the group consisting of the amino acid sequenceof said polypeptide, its nucleotide sequence, and/or the inhibitoraccording to the invention or a fragment thereof as above-described.Said diagnostic device may comprise also necessary reactants and mediafor the diagnostic and/or dosage of the nucleotide and/or amino acidsequence of the polypeptide according to the invention, which are basedupon the method selected from the group consisting of in situhybridisation, hybridisation by labelled antibodies, especially RIA(Radio Immuno Assay) or ELISA (Enzymes Linked Immuno-Sorbent Assay)technologies, detection upon filter, upon solid support, in solution, insandwich, upon gel, dot blot hybridisation, Northern blot hybridisation,Southern blot hybridisation, isotopic or non-isotopic labelling (byimmunofluorescence or biotinilised probes), genetic amplification,(especially by PCR or LCR), double immunodiffusion technique,counter-electrophoresis technique, haemagglutination or a mixturethereof.

Another aspect of the present invention concerns a diagnosis methodwherein a biological sample from the patient, such as cephalo-rachidianfluid, serum, blood, plasma, urine, broncho-alveolar lavage, stomachlavage, etc., is isolated from the patient, and is put in contact withthe diagnostic device according to the invention for the diagnosis orthe monitoring of an injury or a disease, preferably a lung injury or anoxidative stress-related disorder, affected by the presence ofpro-oxidant agent or oxidative stress such as specific cardio-vasculardiseases like arteriosclerosis, neurodegenerative disorders (Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis), apoptosis,inflammatory reactions, allergic reactions such as asthma, hay fever andeczema, high bone mass syndrome, osteopetrosis,osteoporosis-pseudoglioma syndrome, and Bardet-Biedl syndrome 1. Saiddiagnosis and monitoring upon one or more biological samples obtainedfrom several tissues from the patient can be advantageously obtained byone or more of the methods above-described, which could be adaptedaccording to the specific biological sample by the person skilled in theart.

Therefore, the product according to the invention could be used as amarker for the above-identified injuries, diseases or disorders in abroad spectrum of tissues as shown in the enclosed FIG. 1.

A further aspect of the present invention is related to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and anelement selected from the group consisting of the nucleotide sequence,the amino acid sequence of the polypeptide according to the invention,the inhibitor directed against said sequences and/or one or moreportions thereof.

A last aspect of the present invention is related to the use of thepharmaceutical composition according to the invention for themanufacture of a medicament in the treatment and/or the prevention oflung injuries and/or diseases or of oxidative stress-related disorders.

The present invention is also related to a prevention and/or treatmentmethod of a patient, especially a human patient, preferably affected bylung injuries and/or diseases or by oxidative stress-related disorders,wherein a sufficient amount of the pharmaceutical composition accordingto the invention is administered to said patient in order to treat,avoid and/or reduce the symptoms of said injuries and/or diseases.

Other injuries and/or diseases which can be prevented and/or treated areinjuries and/or diseases affected by the presence of pro-oxidant agentsor oxidative stress, such as specific cardio-vascular diseases likearteriosclerosis, neurodegenerative disorders such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, apoptosisand inflammatory reactions and some allergic reactions such as asthma,hay fever and eczema, high bone mass syndrome, osteopetrosis,osteoporosis-pseudoglioma syndrome, and Bardet-Biedl syndrome 1.

The pharmaceutically acceptable carrier according to the invention isany compatible non-toxic substance suitable for administering thecomposition according to the invention to a human patient.Pharmaceutically acceptable carriers according to the invention suitablefor oral administration are the ones well known by the person. skilledin the art, such as tablets, coated or non-coated pills, capsules,spray-gas, patches, gels, solutions or syrups. Pharmaceuticallyacceptable carriers vary according to the mode of administration(intravenous, intramuscular, subcutaneous, parenteral, etc.), and maycomprise also adjuvants well known by the person skilled in the art toincrease, reduce and/or regulate humoral, local and/or cellular responseof the immune system.

The pharmaceutical composition according to the invention may beprepared by the methods, generally applied by the person skilled in theart in the preparation of various pharmaceutical compositions, whereinthe percentage of the active compound/pharmaceutically acceptablecarrier can vary within very large ranges, only limited by the toleranceof the patient to said pharmaceutical composition, and wherein thelimits are particularly determined by the frequency of administrationand the possible side-effects of the active compounds or itspharmaceutically acceptable carrier.

Another aspect of the invention is related to the use of the diagnosticdevice according to the invention for performing upon the patient orupon a biological fluid obtained from the patient, a diagnosis, a dosageand/or a monitoring of the above-mentioned injuries or diseases oroxidative stress-related disorders affecting the patient.

A further aspect of the present invention is related to a cell or anon-human animal, preferably a mammal such as a mouse or a rat,transformed by the vector according to the invention and overexpressingthe polypeptide according to the invention, or a non-human animal,preferably a mammal such as a mouse or a rat, genetically modified by apartial or total deletion of its genomic sequence encoding thepolypeptide according to the invention (knock-out non-human mammal) andobtained by methods well known by the person skilled in the art such asthe one described by Kahn et al. (Cell, Vol. 92, pp. 593-596 (March1998)).

Other examples of genetically modified non-human animals according tothe invention may be a transgenic non-human animal comprising aninhibitor according to the invention, preferably an antisense nucleicacid sequence complementary to the nucleotide sequence according to theinvention so placed as to be transcribed into antisense MRNA which iscomplementary to the nucleotide sequence according to the invention andwhich hybridises to said nucleotide sequence, thereby reducing orblocking its translation.

Further aspects of the present invention will be described in theenclosed non-limiting examples in reference to the following Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a dot blot analysis of mRNA encoding the polypeptideaccording to the invention in various types of human tissues.

FIG. 2 represents a Northern blot analysis of mRNA encoding thepolypeptide according to the invention in a rat lung afteradministration of lipopolysaccharides (LPS) inducing an inflammatoryreaction of the lung.

FIG. 3 represents a Northern blot analysis of mRNA encoding thepolypeptide according to the invention in a rat lung afterintraperitoneal injection of pneumotoxicants.

FIG. 4 is a schematic representation of the human genomic sequence, thecomplete cDNA sequence and the corresponding amino acid sequence.

FIG. 5A-C represents respectively the alignment of the sequences of thehuman B18 polypeptide according to the invention (SEQ ID NO: 2) with thecorresponding rat (SEQ ID NO: 4) and mouse (SEQ ID NO: 6) sequences.

EXAMPLE 1 Homology Between the B18 Polypeptide According to theInvention with Other Known Nucleotide or Amino Acid Sequences

The BLAST 2.0 software (gapped BLAST at the NCBI Internet site) was usedfor searching for homologies between human B18 (162 amino acids) andknown polypeptides in databases (GenBank, SwissProt). Said search didnot give perfect alignment with known peptides from different species(Table 1). Homologies of the human B18 cDNA (805 nucleotides) withGenBank, EMBL, DDBJ and PDB deposited nucleotide sequences (Table 2) andGenBank Expression Sequence TAGS (ESTs) were noted.

TABLE 1 Homologies of the B18 proteins (162 amino acid) with otherproteins Identity (%) Name NCBI ID Homology (%) Membrane protein 165285957/129 (44%) (synechocystis sp.) 81/129 (62%) Peroxisomal-like protein2769700 56/176 (31%) (Aspergillus fumigatus) 90/176 (50%) Haein HI0572hypothetical 1723174 53/146 (36%) protein 80/146 (54%) (Haemophilusinfluenzae) PMP20 (Schizosaccharomyces AJ002536 54/161 (33%) pombe)85/161 (52%) Peroxisomal membrane 130360 59/170 (34%) protein A (PMP 20)89/170 (51%) (Candida boidinii) Peroxisomal membrane 130361 58/170 (34%)protein B (PMP 20) 88/170 (51%) (Candida boidinii) Putative peroxisomal1709682 41/138 (29%) protein PMP from yeast 72/136 (51%) (Saccharomycescerevisiae) Alkylhydroperoxide P26427 36/126 (28%) reductase C22 protein56/126 (45%) (Escherichia coli)

TABLE 2 Name Access NO Identity Human mRNA down-regulated in U82616259/263 (98%) cells infected by adenovirus 5 Human mRNA down-regulatedin U82615 300/321 (93%) cells infected by adenovirus 5

In the Table 2, an identity of 98% has been obtained with the alignmentof 259 nucleotides of CDNA B18, which comprises in its totality 805nucleotides, with 263 nucleotides of U82616 CDNA. A similar identity hasbeen obtained with the U82615 sequence.

The sequence SEQ ID NO 1 comprising 805 nucleotides presents a homologywith several EST sequences obtained from a human and from a mouse,having the following references:

Human

AA130751, N42215, W38597, N91311, N68467, AA187737, N68916, W00593,R88950, AA181884, H20154, H66666

Mouse

AA220019, AA123351, AA087129, AA255021, AA249897, W71344

EXAMPLE 2 Tissue Detection

A human RNA master Blot (Clontech) containing 100-500 ng of poly-A+humanRNA in each dot (normalised to the mRNA expression levels of eightdifferent housekeeping genes) was hybridised with a 554 bp-long B18probe labelled with ³²P, and quantified using PhosphorimagingTechnology. As shown in FIG. 1, B18 mRNA is present in all tissuesexamined but predominantly in trachea, lung, kidney, thyroid gland,stomach, colon, heart and some regions of the brain. Highest expressionhas been noted in the thyroid tissue. This presence is probablycorrelated with the possible antioxidant activity of the B18polypeptideaccording to the invention.

EXAMPLE 3 Inflammatory Reaction

FIG. 2 represents a Northern blot analysis of rat lung mRNA after 6, 48and 72 hours after lipopolysaccharides (LPS) instillation inducing aninflammatory reaction in the lung.

A Northern blot containing 15 μg of total RNA in each lane washybridised with a 225 bp-long rat B18 probe, stripped and reprobed witha 572 bp-long rat β-actin probe, both labelled with ³²P. Northern blotwas quantified using Phosphorimaging Technology and the B18 mRNA datawere normalised to β-actin mRNA level.

EXAMPLE 4 Pneumotoxic Reaction

FIG. 3 represents a Northern blot analysis of rat lung mRNA afterintraperitoneal injection of pneumotoxicants (4-ipomeanol,1-(3-fyryl)-4-hydroxypentanone (IPO), methylcyclopentadienyl manganesetricarbonyl (MMT) or alpha naphtylthiourea (ANTU)). These agents areknown to induce in the lung acute lesions of Clara (IPO) and alveolarcells (MMT) as well as increasing the permeability of the alveolar/bloodbarrier (ANTU). A Northern blot containing 15 μg of total RNA in eachlane was hybridised with a 225 bp-long rat B18 probe, stripped andreprobed with a 572 bp-long β-actin probe both labelled with ³²P. TheNorthern blot was quantified using Phosphorimaging Technology and ratB18 mRNA data were normalised to β-actin mRNA level.

EXAMPLE 5 Proteinic Activity of the B18 Polypeptide

An amino analysis of the complete human B18 amino acid sequence showsthat said polypeptide presents specific portions showing a homology withother antioxidant enzymes (starting from a Leucine at position 37 untila Cysteine at position 48) and another portion having an importanthomology with beta chains of ATP synthase (starting from a Glutamic acidat position 14 until a Glycine in position 39).

Furthermore, the B18 amino acid sequence according to the inventionshows an important homology with an Aspergillus fumigatus allergen (34%identity and 60% homology by using clustal V sequence alignment),especially upon the portion of said B18 polypeptide having possibleantioxidant properties. Therefore, it is possible that a peroxisomalprotein (possibly homologous to B18 protein) is able to induce and tobind IgE from patients sensitised to Aspergillus fumigatus peroxisomalproteins after an induction of the patient immune system withAspergillus fumigatus allergen. This mechanism can be compared to areaction obtained with the manganese superoxide dismutase (MnSOD)wherein the human MnSOD is able to bind to IgE from patients sensitisedto Aspergillus fumigatus MnSOD.

Furthermore, the Inventors have identified a position of the B18polypeptide which presents a homology with a Cyclophilin-binding domainof Candida boidinii PMP20 (receptor of the immuno-suppresant drugcyclosporine A). Said possible Cyclophilin-binding domain is startingfrom the Threonine in position 151 until the Leucine in position 162.

EXAMPLE 6: B18 Human Gene and mRNA Alternative Splicing

As represented in the enclosed FIG. 4, the Inventors have identifiedupon the genomic DNA (SEQ ID NO: 10) 5 exons and 5 introns. By RT-PCR(using primers 5′-gggtatgggactagctggcg-3′ (SEQ ID NO: 15) and5′-ctggccaacattccaattgcag-3′(SEQ ID NO: 16)) and according to thegenomic sequence, 4 different cDNAs corresponding to the transcriptionof the said genomic DNA have been identified in human lung and in humanbrain. A first cDNA of 736 bp corresponds to the cDNA encoding thecomplete amino acid sequence of the B18 protein according to theinvention. However, 3 other cDNAs of 601, 604, and 469 bp were alsoidentified, and comprise specific splicings of one of more exons.

Therefore, another aspect of the present invention is related to saidspecific portions of the complete genomic or CDNA nucleotide sequenceaccording to the invention or to specific portions of the complete aminoacid sequence of the B18 protein according to the invention, which couldbe used also as specific markers of the B18 activity, preferably theanti-oxidative activity.

EXAMPLE 7 Knock-out Mouse

Exons of a mouse genomic sequence encoding the B18 polypeptide accordingto the invention have been deleted by homologous recombination. Saidhomologous recombination has been obtained with a genetic sequencecomprising a neomycin resistant gene. The targeting vector with saidgene and a thymidine kinase (in order to eliminate non-homologousrecombinants with ganciclovir) has been prepared. Said recombination wasused for the deletion of one or more exons of the B18 polypeptide. Afterelectroporation of ES cells with the targeting vector, positive cloneshaving incorporated homologous recombination were identified by Southernblot with labelled probes. Aggregation of said positive clones with amorula from a Swiss pseudo-pregnant mouse produces several chimeric micewhich survive after birth. Several homozygote mice are obtained bycross-breeding and are used as a model for the above-mentioned diseases.

Similar experiments may be done with another mammal whose B18 sequenceis known (the B18 sequence of a mouse and a rat and their alignment withthe human sequence is shown in the enclosed FIG. 5).

EXAMPLE 8 Chromosome Localisition of Human B18 Gene

Radiation hybrid clones (GeneBridge 4 Radiation Hybrid Panel, ResearchGenetics) were used for performing chromosome localisation by PCR withtwo different pairs of primers (5′-caggttcaccttgttccctggctc-3′ (SEQ IDNO 14), 5′-atgttatgcaaccctttgcgacac-3′ (SEQ ID NO 17) and5′-gtgtttgaaggggagccagggaac-3′ (SEQ ID NO 18),5′-agagacagggtttcaccatcttgg-3′ (SEQ ID NO 19)).

The Inventors have located B18 genomic sequence on human chromosome11q13. B18 gene has been located 7.15-6.1 cR from marker D11S913 betweenmarkers D11S1963 and D11S4407 (Genome Database internet site).

Unknown genes linked to different disorders have been localised in thesame region of chromosome 11. Therefore, B18 gene is possibly associatedwith these disorders:

atopy (atopic hypersensitivity: asthma, hay fever and eczema; MIM °No.147050 at OMIM of NCBI internet site),

high bone mass syndrome (MIM °No. 601884),

osteoporosis (MIM °No. 259700),

osteoporosis-pseudoglioma syndrome (MIM °No.601884) and

Bardet-Biedl syndrome 1(MIM °No. 209901).

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 4 <210> SEQ ID NO 1 <211>LENGTH: 805 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (193)..(681) <400> SEQUENCE: 1gccaggaggc ggagtggaag tggccgtggg gcgggtatgg gactagctgg cgtgtgcgcc 60ctgagacgct cagcgggcta tatactcgtc ggtggggccg gcggtcagtc tgcggcagcg 120gcagcaagac ggtgcagtga aggagagtgg gcgtctggcg gggtccgcag tttcagcaga 180gccgctgcag cc atg gcc cca atc aag gtg gga gat gcc atc cca gca gtg 231Met Ala Pro Ile Lys Val Gly Asp Ala Ile Pro Ala Val 1 5 10 gag gtg tttgaa ggg gag cca ggg aac aag gtg aac ctg gca gag ctg 279 Glu Val Phe GluGly Glu Pro Gly Asn Lys Val Asn Leu Ala Glu Leu 15 20 25 ttc aag ggc aagaag ggt gtg ctg ttt gga gtt cct ggg gcc ttc acc 327 Phe Lys Gly Lys LysGly Val Leu Phe Gly Val Pro Gly Ala Phe Thr 30 35 40 45 cct gga tgt tccaag aca cac ctg cca ggg ttt gtg gag cag gct gag 375 Pro Gly Cys Ser LysThr His Leu Pro Gly Phe Val Glu Gln Ala Glu 50 55 60 gct ctg aag gcc aaggga gtc cag gtg gtg gcc tgt ctg agt gtt aat 423 Ala Leu Lys Ala Lys GlyVal Gln Val Val Ala Cys Leu Ser Val Asn 65 70 75 gat gcc ttt gtg act ggcgag tgg ggc cga gcc cac aag gcg gaa ggc 471 Asp Ala Phe Val Thr Gly GluTrp Gly Arg Ala His Lys Ala Glu Gly 80 85 90 aag gtt cgg ctc ctg gct gatccc act ggg gcc ttt ggg aag gag aca 519 Lys Val Arg Leu Leu Ala Asp ProThr Gly Ala Phe Gly Lys Glu Thr 95 100 105 gac tta tta cta gat gat tcgctg gtg tcc atc ttt ggg aat cga cgt 567 Asp Leu Leu Leu Asp Asp Ser LeuVal Ser Ile Phe Gly Asn Arg Arg 110 115 120 125 ctc aag agg ttc tcc atggtg gta cag gat ggc ata gtg aag gcc ctg 615 Leu Lys Arg Phe Ser Met ValVal Gln Asp Gly Ile Val Lys Ala Leu 130 135 140 aat gtg gaa cca gat ggcaca ggc ctc acc tgc agc ctg gca ccc aat 663 Asn Val Glu Pro Asp Gly ThrGly Leu Thr Cys Ser Leu Ala Pro Asn 145 150 155 atc atc tca cag ctc tgaggccctgggc cagattactt cctccacccc 711 Ile Ile Ser Gln Leu 160 tccctatctcacctgcccag ccctgtgctg gggccctgca attggaatgt tggccagatt 771 tctgcaataaacacttgtgg tttgcggaaa aaaa 805 <210> SEQ ID NO 2 <211> LENGTH: 162 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met Ala Pro IleLys Val Gly Asp Ala Ile Pro Ala Val Glu Val Phe 1 5 10 15 Glu Gly GluPro Gly Asn Lys Val Asn Leu Ala Glu Leu Phe Lys Gly 20 25 30 Lys Lys GlyVal Leu Phe Gly Val Pro Gly Ala Phe Thr Pro Gly Cys 35 40 45 Ser Lys ThrHis Leu Pro Gly Phe Val Glu Gln Ala Glu Ala Leu Lys 50 55 60 Ala Lys GlyVal Gln Val Val Ala Cys Leu Ser Val Asn Asp Ala Phe 65 70 75 80 Val ThrGly Glu Trp Gly Arg Ala His Lys Ala Glu Gly Lys Val Arg 85 90 95 Leu LeuAla Asp Pro Thr Gly Ala Phe Gly Lys Glu Thr Asp Leu Leu 100 105 110 LeuAsp Asp Ser Leu Val Ser Ile Phe Gly Asn Arg Arg Leu Lys Arg 115 120 125Phe Ser Met Val Val Gln Asp Gly Ile Val Lys Ala Leu Asn Val Glu 130 135140 Pro Asp Gly Thr Gly Leu Thr Cys Ser Leu Ala Pro Asn Ile Ile Ser 145150 155 160 Gln Leu <210> SEQ ID NO 3 <211> LENGTH: 780 <212> TYPE: DNA<213> ORGANISM: Rattus rattus <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (136)..(624) <221> NAME/KEY: Unsure <222> LOCATION:(136)..(624) <223> OTHER INFORMATION: purine <221> NAME/KEY: Unsure<222> LOCATION: (323)..(323) <223> OTHER INFORMATION: pyrimidine <221>NAME/KEY: Unsure <222> LOCATION: (371)..(371) <223> OTHER INFORMATION:pyrimidine <400> SEQUENCE: 3 tgcgtcctag gcagcatagc cggatcggtg ctccgtgcatcggctacttg gacgtgcgtg 60 gcaggcagag caggccggaa aggagcaggt tgggagtgtggtggggcccg cagcttcagc 120 agtgccgcgg tgact atg gcc ccg atc aag gtg ggagac acc att ccc tca 171 Met Ala Pro Ile Lys Val Gly Asp Thr Ile Pro Ser1 5 10 gtg gag gta ttt gra ggg gaa cct gga aag aag gtg aac ttg gca gag219 Val Glu Val Phe Xaa Gly Glu Pro Gly Lys Lys Val Asn Leu Ala Glu 1520 25 ctg ttc aag gac aag aaa ggt gtt ttg ttt gga gtc cct ggg gca ttt267 Leu Phe Lys Asp Lys Lys Gly Val Leu Phe Gly Val Pro Gly Ala Phe 3035 40 aca cct ggc tgt tcc aag acc cat ctg cct ggg ttt gtg gag caa gcc315 Thr Pro Gly Cys Ser Lys Thr His Leu Pro Gly Phe Val Glu Gln Ala 4550 55 60 gga gct cyg aag gcc aag gga gca caa gtg gtg gcc tgt ctg agt gtt363 Gly Ala Xaa Lys Ala Lys Gly Ala Gln Val Val Ala Cys Leu Ser Val 6570 75 aat gat gyc ttc gtg act gca gag tgg ggt cga gcc cac cag gca gaa411 Asn Asp Xaa Phe Val Thr Ala Glu Trp Gly Arg Ala His Gln Ala Glu 8085 90 ggc aag gtt cag ctc ctg gct gac ccc act gga gct ttt gga aag gag459 Gly Lys Val Gln Leu Leu Ala Asp Pro Thr Gly Ala Phe Gly Lys Glu 95100 105 aca gat tta cta cta gat gat tct ttg gtg tct ctc ttt ggg aat cgt507 Thr Asp Leu Leu Leu Asp Asp Ser Leu Val Ser Leu Phe Gly Asn Arg 110115 120 cgg cta aaa agg ttc tcc atg gtg ata gac aag ggc gta gta aag gca555 Arg Leu Lys Arg Phe Ser Met Val Ile Asp Lys Gly Val Val Lys Ala 125130 135 140 ctg aac gtg gag ccg gat ggc aca ggc ctc acc tgc agc ctg gccccc 603 Leu Asn Val Glu Pro Asp Gly Thr Gly Leu Thr Cys Ser Leu Ala Pro145 150 155 aac atc ctc tca caa ctc tga ggccctgacc agaatgtcct ctgactctcc654 Asn Ile Leu Ser Gln Leu 160 catctcctcc acccagctct gggccaaaggcccagtacct ccttacctga gggccactgg 714 aatggaacct tgacaatatt tctgcaataaacagtttaat ttgtgaaaaa aaaaaaaaaa 774 aaaaaa 780 <210> SEQ ID NO 4 <211>LENGTH: 162 <212> TYPE: PRT <213> ORGANISM: Rattus rattus <220> FEATURE:<221> NAME/KEY: Modified-site <222> LOCATION: 17 <223> OTHERINFORMATION: Glu or Gly <221> NAME/KEY: Modified-site <222> LOCATION: 63<223> OTHER INFORMATION: Leu or Pro <221> NAME/KEY: Modified-site <222>LOCATION: 79 <223> OTHER INFORMATION: Ala or Val <400> SEQUENCE: 4 MetAla Pro Ile Lys Val Gly Asp Thr Ile Pro Ser Val Glu Val Phe 1 5 10 15Xaa Gly Glu Pro Gly Lys Lys Val Asn Leu Ala Glu Leu Phe Lys Asp 20 25 30Lys Lys Gly Val Leu Phe Gly Val Pro Gly Ala Phe Thr Pro Gly Cys 35 40 45Ser Lys Thr His Leu Pro Gly Phe Val Glu Gln Ala Gly Ala Xaa Lys 50 55 60Ala Lys Gly Ala Gln Val Val Ala Cys Leu Ser Val Asn Asp Xaa Phe 65 70 7580 Val Thr Ala Glu Trp Gly Arg Ala His Gln Ala Glu Gly Lys Val Gln 85 9095 Leu Leu Ala Asp Pro Thr Gly Ala Phe Gly Lys Glu Thr Asp Leu Leu 100105 110 Leu Asp Asp Ser Leu Val Ser Leu Phe Gly Asn Arg Arg Leu Lys Arg115 120 125 Phe Ser Met Val Ile Asp Lys Gly Val Val Lys Ala Leu Asn ValGlu 130 135 140 Pro Asp Gly Thr Gly Leu Thr Cys Ser Leu Ala Pro Asn IleLeu Ser 145 150 155 160 Gln Leu

What is claimed is:
 1. An isolated or purified polynucleotide consisting of SEQ ID NO: 1 or its complementary strand.
 2. A vector comprising the polynucleotide of claim
 1. 3. A diagnostic device comprising the polynucleotide of claim
 1. 4. An isolated cell transformed by the vector according to claim
 2. 